The currents through individual channels located in the membranes of skeletal and cardiac muscle will be measured on fragments of membrane which have been excised from their cells of origin. The excised membrane technique permits direct experimental control of the transmembrane potential and the solutions bathing both sides of the membrane. It is sensitive enough to measure the currents which pass through individual channels. This technique will be used to investigate three specific problems during the proposed project period: 1) Measure the kinetics of Na channels in tissue cultured rat skeletal muscle after removal of their inactivation with N-bromoacetamide or pronase. These agents must be applied from the membrane's cytoplasmic side. Removal of inactivation and single channel recording permit the determination of a wider range of kinetic parameters than other techniques. Control of all solutions also allows drug application and ionic gradient reversal, thereby permitting channel studies over a broad range of potentials. 2) Identify and describe the Ca-dependent K channel in cardiac Purkinje fibers. This channel is important for regulation of the cardiac action potential and cardiac drug responses. Studies will involve application of buffered and iontophoretic CA++ to the membrane's cytoplasmic surface. The channel's conductance, kinetics and CA++ binding properties will be investigated. 3) Determine the mechanism of the ACh induced increase in K-permeability in cardiac Purkinje fibers. CA++ and cyclic-GMP have been proposed as "second messengers" in this system. Both of these substances will be controlled on the membrane's cytoplasmic surface in order to study their interrelationship and target of action. The hypothesis that CA++ acts to regulate the Ca-activated K conductance will be investigated. My long term goal is to develop the methodology for studying membrane channel biophysics in heart and other difficult-to-study tissues with the precision common to present day squid axon studies. The importance of this goal, and of the specific studies proposed here is that accurate and detailed understanding of membrane physiology is necessary for rational drug development and administration in the fields of Cardiovascular and Neural Medicine.